The recent interest in nano-scale chromatography (<1 μL/min flow rates) has prompted HPLC instrument manufacturers to try to develop pumps capable of delivering lower flow rates. Unfortunately, typical analytical-scale HPLC pump technology does not scale well to these low flow rates as the constant-flow open-loop analytical-scale pumps typically used for analytical-scale chromatography (0.1-5 mL/min) are good flow sources above ˜0.1 μL/min, but below these flow rates, inaccuracies due to solvent compression and seal, fitting or check-valve leakage compromise their flow accuracy.
Traditional plunger displacement pumping systems have been successful in delivering stable, accurate flows in the normal-scale and micro-scale high performance liquid chromatography regimes. While normal-scale HPLC is performed with mobile phase flow rates of about 0.1-5.0 mL/min and micro-scale HPLC is performed with mobile phase flow rates of about 1-100 μL/min, nano-scale HPLC requires mobile phase flow rates in the 50-1000 nL/min range. Current plunger displacement pumping systems typically cannot deliver nano-scale HPLC flow rates with reliability and accuracy.
One method for providing nano-scale flow rates in an HPLC system is to use a flow-divider which directs a majority of flow from the pump to a waste stream and a small portion of the pump output to the HPLC working stream (i.e., to the liquid chromatography column). A split restrictor in the waste stream and/or the working stream controls the split ratio of the system. Normal-scale or micro-scale HPLC pumps can be used in split flow mode to produce nano-scale HPLC flow rates in the working stream.
Unfortunately, in order to operate a HPLC system in split-flow mode the user must calculate the split ratio of the system. To calculate the split ratio, the user must know the permeabilities of both the split restrictor and the chromatographic system (i.e. the packed column). These permeabilities are used to calculate the flow rate that must be supplied by the normal-scale or micro-scale HPLC pump to produce the desired flow through the chromatographic system. Although it is possible to calculate split restrictor dimensions that should provide a desired split ratio, changes in permeability of either the split restrictor or chromatographic column over time cause unpredictable split ratio variations. Such variations result in unacceptable flow variations through the chromatographic column.
One possible solution to the problem of changing split ratios is to monitor the flow to the chromatographic column with an appropriate flow sensor. Fluid flow rates can be determined by measuring the pressure of a liquid flowing through a restrictor. Assuming a constant viscosity, the back pressure of liquid flowing through a restrictor will scale linearly with the flow rate of the liquid. The flow rate is measured by placing a pressure transducer before and after a restrictor inline with the flow. Signals from the pressure transducers are electronically subtracted and amplified to achieve a high degree of common-mode noise rejection.
The permeability of the restrictor is chosen so that it provides sufficient back pressure to produce a measurable pressure difference signal (ΔP) in the flow ranges of interest but does not produce a significant back pressure for the pump. For example, a 10 cm long, 25 μm inside diameter capillary will provide a back pressure of approximately 100 pounds per square inch (psi) for water flowing at 5 μL/min. This permeability is sufficient for providing a flow measurement while not inducing much fluidic load on the pump.
However, pressure measuring flow sensors must be calibrated to compensate for the different viscosity of each fluid being measured. This creates a great disadvantage in liquid chromatography applications wherein fluid composition varies dramatically over the course of a chromatography run.
Another method that can be used to sense fluid flow is thermal flow sensing. Several companies including Sensirion AG, of Zurich, Switzerland, and Bronkhorst Nijverheidsstraat of Ruurlo, The Netherlands, have been developing thermal flow sensors capable of monitoring flows in nL/min ranges.
In the operation of these thermal flow sensors, heat introduced into a liquid filled tube/channel will disperse in both the upstream and downstream directions (i.e. due to thermal conduction or diffusion respectively). The tube of the flow sensing device is made from materials of low thermal conductivity (i.e. glass, plastic). A temperature profile will develop when a discrete section of the fluid in the tube is continuously heated, under a zero flow condition. The shape of this temperature profile will depend upon the amount of heat added to the fluid and the upstream and downstream temperatures of the liquid. Assuming identical upstream and downstream fluid temperatures, under a zero-flow condition, liquid temperatures measured at first and second sensor will be equal as thermal diffusion will be equal in both directions.
If the liquid in the tube is permitted to flow, the fluid temperatures at the first and second sensor will depend upon the rate of liquid flux and the resulting heat convection. As liquid begins to flow past the heated zone, a temperature profile develops. In addition to the symmetric diffusion of the heat, asymmetric convection of the heated fluid will occur in the direction of the fluid flow. Therefore, under flowing conditions, fluid temperatures measured at the first and second sensor will be different.
Temperature measurements made at the first and second sensor are sampled, subtracted and amplified electronically in situ to provide a high degree of common-mode noise rejection. This allows discrimination of extremely small upstream and downstream temperature differences. By appropriate placement of temperature measurement probes (i.e., first and second sensor) and/or by changing the amount of heat added to the flowing liquid, temperature measurement can be made at inflection points along the temperature profile. Measurement at the inflection points maximizes the upstream/downstream ΔT response to flow rate change.
However, like pressure measuring flow sensors which must be calibrated to compensate for the different viscosity of each fluid being measured. Thermal based flow sensors also need such calibration. This at times creates a disadvantage in liquid chromatography applications wherein fluid composition varies dramatically over the course of a chromatography run.
Other pump solutions for creating the low flow rates required by nano-scale LC involve single-stroke syringe pumps. These pumps have a fixed delivery volume. As a result run times may be limited by the length of the pump stroke. Time is required between each run to refill the pump. During this refill cycle, the chromatographic system must depressurize, then re-pressurize to start the next run. Repeated depressurization/re-pressurization cycles unfortunately have a deleterious effect on the chromatographic column.
Additionally, Nano-scale LC systems are often coupled to mass spectrometers. Electro-spray interfaces typically used in LC-coupled mass spectrometers are most stable when constantly flowing. The stop-flow conditions existing during refill cycles of syringe-type pumps as noted above may destabilize the electro-spray mass spectrometry interface.